DE3333752A1 - PHARMACOLOGICALLY ACTIVE PEPTIDES - Google Patents

Abstract

PCT No. PCT/DE84/00191 Sec. 371 Date May 13, 1985 Sec. 102(e) Date May 13, 1985 PCT Filed Sep. 13, 1984 PCT Pub. No. WO85/01292 PCT Pub. Date Mar. 28, 1985.Pharmacologically active peptides of the following formulas are described: L-Tyr-X-L-Phe-X-T L-Tyr-X-L-Phe-X-A-T L-Tyr-X-L-Phe-X-A-X-T L-Tyr-X-L-Phe-X-A-X-B-T in which L-Tyr is the N-terminal amino acid residue L-tyrosine, X any amino acid residue of the D-form, L-Phe is the amino acid L-phenylalanine, A and B are any desired amino acid residues and T is the C-terminal carboxyl group, which is further disclosed in the description. The substances are suitable for the therapy of diseases.

Description

■ ίο

14.9.83

Our reference Cas S / 83 Int.Cl.

C 07 C 103/52 A 61 K 37/02 C 07 G - 7/00

Applicant:

Dipl. Chem. Victor Brantl ■ Frauenplatz 10 8000 Munich 2

PHARMACOLOGICALLY ACTIVE PEPTIDES

description

From DT-OS 29 36 099 are pharmacological. active peptides known, which in particular act opiate-like, which in comparison to the peptides of the. DT-OS 29 21 216 stronger opiate-like Trigger effects and a higher stability towards proteolytic enzymes (e.g. im Rat plasma). The peptides of DT-OS 29 36 099 have the following basic structure

1 ? 3 4
L-Tyr - X -L-Phe -L-Pro -

where L-Tyr = L-tyrosine, X is a D-amino acid; L-Phe = L-phenylalanine and L-Pro = L-proline. It should be noted at this point that the 4th amino acid according to DT-OS 29 36 099 is in the L-form ; the D-shape is not mentioned. It has been shown that these peptides only have a certain maximum potency, in particular an opiate-like one (see Table 1, p. 20)

DT-OS 30 34 897, which is very similar to DT-OS 29 36 099, describes pharmacologically active peptides of the following basic structure:

L-Tyr-A-L-Phe-B-

where L-Tyr = L-tyrosine, A is a D-amino acid., L-Phe = L-phenylalanine and B is a neutral L-amino acid residue (e.g. glycine). It should also be noted here that the 4th amino acid residue should be in the L shape.

"T

It should be noted at this point that the peptides of the Europ. Registration no. 81305519.1 (Publ. No. 0053029) essentially ident. with those the previously mentioned DT-OS 2921216 are. The Europ. Registration 81305519.1. Owns the position of the 4th amino acid residue of the peptide a D-amino acid (D-Pro), but it was not recognized as having a particularly potent opiate-like effect by the combination of a D-alanine residue in the second position together with a D-proline residue is achieved in the 4th position; "In addition, the peptides of the European application 8130519.1 the disadvantage that these are broken down very quickly in the blood and are therefore pharmacological only for a short time Show effects on living organism. (This degradation is due to the introduction of the D-alanine residue prevented in the second position) the exact data can be found in DT-OS 2921216 and DT-OS 2936099 in particular.

The object of the present invention is to create novel pharmacologically active peptides, especially opiate-like, the stronger pharmacological effects, especially opiate-like and / or have higher enzymatic stability compared to peptide-cleaving enzymes.

According to the invention, this object is achieved by that the peptides have the following structure:

L-Tyr-XL-Phe-XT
L-Tyr-XL-Phe-XAT
L-Tyr-XL-Phe-XAXTL-Tyr-XL-Phe-XAXBT

where L-Tyr for the N-terminal amino acid residue L-tyrosine, X for any amino acid residue of the D-configuration, L-Phe for the amino acid residue L-phenylalanine. ' A and B can be any amino acid residues - T stands for OH, QR, NH ₂ , NHR, NR ₂ or NHNHR ¹ , where R optionally has the following meaning: Substib. linear or branched C. _1Q -alkyl, adamantyl, C. C, _ß- aralkyl, C ~ "-alkenyl, linear, branched or cyclic aliphatic. C.. ^ - acyl, optionally substituted by OH, NH_, C.. -Alkoxy or halogen, aromatic acyl, optionally substituted by OH, NH-, halogen or C ..- alkoxy; C 1-6 linear branched or cyclic aliphatic urethane or aromatic urethane and their pharmaceutically acceptable salts.

The peptides according to the invention show in contrast to the peptide described in the prior art a stronger opiate-like effect and / or ο i nc: er

increased stability in relation to peptide-cleaving enzymes (see exemplary embodiment).

According to a development of the invention are
the peptides characterized; that the
Aminic acid residue X through the amino acids:

-D-alenine, D-threonine, D-serine, D-methionine, D-valine, D-phenylalanine, D-leucine, D-isoleucine, D-arginine, D-histamine, D-proline, D-hydroxyproline, D-lysine, D-glutamine, D-glutamic acid, D-asparagine, D-aspartic acid and A, = B by the aromatic amino acids tyrosine or phenylalanine, or glycine is or are replaced.

The special properties of the peptides according to the invention are presumably wholly or partly due to the special arrangement of the D-amino acid residues. These are in particular those in the 2nd position (starting from the tyrosine) and in the 4th position which
the high enzyme. Condition stability and high opiate-like effectiveness. (See embodiment)

According to another development, the peptides are characterized by this, three of these (in; N-terrainale Tyrosine of the formula:

have, where in detail mean:

R, for hydrogen or an alkyl group with 1

up to 4 carbon atoms,
R. for hydrogen or together with R, for

an ethylene bridge,
Rc for hydrogen, an alkyl group with 1 to C atoms or an R ^ CO group,

R, - for a saturated or unsaturated, straight chain or branched alkyl radical with 1 to 17 carbon atoms, a phenyl radical or a Phenylalkyl radical with 7 to 12 carbon atoms, the phenyl radicals having 1 or 2 substituents substituted from the series halogen, alkyl with 1 to 4 carbon atoms or alkoxy with 1 to 4 carbon atoms can be, where the R <-0 group in meta- or para-position to the

-CH ₂ -C-CO-ReSt is located,

NHW

W for hydrogen, alkyl with 1 to 5 carbon atoms, alkenyl with 3 to 5 carbon atoms, cyclopropylmethyl, Cyclobuty! Methyl, R, CO-,

b) the phenylalanine in the general formula:

is present, where in detail:

R ₇ for hydrogen or alkyl with 1 to 4 carbon atoms ,

R ₀ for hydrogen, fluorine, chlorine, bromine, nitro, ο

Alkyl with 1 to 4 carbon atoms, ζ for 1 or 2 stands.

This further development has the advantage that, for example, by introducing halogens such as Cl or F in the arom. Ring of the phenylalanine the lipophilicity of the substances is increased so that they get better from the blood to the central one Nervous system and so less substance for the same effect is needed. Likewise affects the introduction of an N-Me thy 1 group at Pheny la lan inres t au3.

The peptides according to the invention can after a Further be characterized in that the amino acid residues: alanine, phenylalanine and A and B exist as, dehydroamino acids.

To further increase the enzymat: stability can the phenylal in the 3rd position of the peptide are in the D-form. Surprisingly it has this does not result in a loss of the opiate-like effect and leads to an even higher enzymatic effect Stability of the peptides.

The peptides according to the invention show strong Effects on the central nervous system. This can in particular strong opiate-like (analgesic) Effects as well as cataleptic effects. Effects can also be observed which can be described as neuroleptic-like.

The peptides according to the invention also have Effects on the cardiovascular system. This can especially be an antihypertensive one Be effect.

The peptides according to the invention can be in different Forms are administered (e.g. tablets, or in solution); the type of application can be oral or parenteral (e.g. i.v., i.m. and s. c.), transdermal, nasal, vaginal or be rectal. It can be in the different Forms of preparation may contain auxiliaries, dio favor absorption.

The dosage of the active ingredients of a pharmaceutical preparation can according to the different Mode of administration vary widely. .. · Generally, a dose range of 0.001 to mg of active ingredient per kilogram of body weight which must be supplied to the mammalian organism in order to have a therapeutic effect, such as e.g. to achieve analgesia.

According to a particularly advantageous embodiment of the invention, the peptides according to the invention have peptides the following formulas:

TyΓ-0-Ala-Phe-D-Ala ^ TyT-NH ₂ Ty'r-D-Ala.-Phe-D-Ala-Phe-NH ₂ .

• Tyr-D-Ala-Phe-D-Ala-Tyr-D-Pro-NH Ty rD-Ala-P.he-D-Ala-Phe-DP ro-NH ₂ TyT-D-AIa-PhB-D- AIa-TyT-D-PrO-SeT-NH ₂ Tyr-D-Ala-Phe-D-Ala-Phe-D-Pro-Ser-NH ₂ Tyr-D-Ala-Phe-D-Ala-Tyr-D- AIa-Ser-NH ₂ Tyr-D-Ala-Phe-Dr-Ala-Phe-D-Ala-Ser-NH ₂ Tyr-D-Ser-Phe-D-Ala-Tyr-NH ₂ Tyr-D-Ser- Phe-D-Ala-Phe-NH ₂

-Away'

Execution example

First, the synthesis of two is more in accordance with the invention Peptides described by way of example, then the pharmacological properties of these peptides were discussed.

Synthesis of two inventive peptides:

The synthesis of the two peptides:

Tyr-D-Ala-Phe-D-Ala-Tyr-NH ₂

Tyr-D-Ala-Phe-D-Ala-Phe-NH ₂

took place as described by Lottspeich et al. in Hoppe Seylers Z. Physiol. Chem., ..3JkI, 1835-1839 (1980) and in der'DT-OS 29 36 099 was described. Briefly on the synthesis steps received.

The individual amino acid building blocks or the Dipeptiti come from Bachern AG, Bubendorf, Switzerland or from Fluka, Buchs, Switzerland. Level I.

a. Formation of the mixed anhydride of Z-D-AIa

335 mg ZD-Ala (1.5 mmol), Z = Benzoxycarbonylschutzgruppe, are dissolved in 15 ml .Dimethylformamid (DMF) under addition of N-methylmorpholine and reacted with 180 μ1 (1.4 mmol) of isobutyl chloroformate at -15 C ₁ 15 minutes.

b. Preparation of the amino component

195 mg (1.0 mmol) of tyrosine methyl ester * are in 20 ml OMF with the addition of 110 μΐ (1.0 mmol) N-methylmorpholine dissolved at -15 ° C.

After the amino component has been prepared, it becomes to I a. added and for 12 hours at the o.g. Temperature held.

Before work-up, the 50% destroyed at ÜberschuQ is mixed anhydride: At ^{0 °} C, the pH of the reaction product with an aqueous saturated KHCO, solution to pH 8 is set and stirred for 30 min at 0 ° C.

The dipeptide is then extracted with 50-100 ml of ethyl acetate (Etac); the Etac / peptide mixture becomes 5 χ washed with 20 ml of saturated aqueous sodium chloride solution. After two more washes the Etac phase is evaporated with 10 ml of water.

The protective group is split off by hydrogenation; this is the dipeptide in 30 ml Dissolved methanol and added 100 mg of palladium on activated carbon (Merck, Darmstadt). After repressing the air through nitrogen is introduced into the reaction vessel hydrogen. The hydrogenation is carried out at 25-30 C. The hydrogenation is finished when in aqueous barium hydroxide solution no more precipitate is formed. The solution is filtered, washed with water

* or 170 mg phenylanine methylity

wash and rotate on a rotary evaporator. The intermediate product that remains is then used in turn as an amino component in the next coupling step.
Stage II

a. Formation of the mixed anhydride of Z-D-Ala-Phe

600 ₍ mg (1.6 mmol) ZD-Ala-Phe are treated as described under stage I a.

b. Preparation of the amino component

The dipeptide D-Ala-Tyr-OMet obtained in stage I or. The D-Ala-Phe-OMet obtained analogously is as under I b. described dissolved and · with the mixed anhydride of stage II a. implemented (12 hours at -15 ⁰ C).

The work-up, extraction and hydrogenation are carried out as described in stage I.

The end product of this stage is the tetrapeptide D-Ala.-Phe-D-Ala-Tyr-OMet or D-Ala-Phe-D-Ala-Phe-OMet obtain.

Stage III

a. Formation of the mixed anhydride of Z-Tyr

629 mg (1.4 mmol) Z-Tyr, 160 μΐ (1.4 mmol) and 170 μΐ (1.3 mmol) isobutyl chloroformate are dissolved in 15 ml of DMF as previously described

b. Preparation of the amino component

The stage II tetrapeptides (see above) are dissolved in 15 ml of DMF and mixed with the anhydride level III a. implemented. The rest of the procedure and work-up are carried out as before described in detail above.

Stage IV /

After the protective group has been split off, the peptide methyl esters undergo ammonolysis in the usual way subject to convert these into the corresponding amides.

step \ l

The peptides are purified after ammonolysis, as described by Lottspeich et al. in Hoppe Seylers Z. Physiol. Chem., 361 , p. 1836 "(1980). The end product of the synthesis is purified by gel chromatography and then passed on to the amino acid analysis.

The amino acid analysis corresponded to the composition of the peptides according to the invention:

Tyr-D-Ala-Phe-D-Ala-Tyr-NH Tyr-D-Ala-Phe-D-AIa-Phe-NH ₂

The following part is about the pharmacological Properties of these two exemplary embodiments according to the invention Peptides reported.

Analogous to the procedure described above, you can further peptides according to the invention of the following formulas can be obtained, such as, for example:

Tyr-D-Ala-Phe-D-Ala-Tyr-NH ₂ Tyr-D-Ala-PhC-D-AIa-PhC-NH ₂ TyT-D-AIa-PhB-D-AIa-TyT-D-PrO- NH ₂

TyT-D-AIa-PhB-D-AIa-TyT-D-PrO-SeT-NH ₂ 'Ty _r _D-Ala-Phe-D-Ala-Phe-D-Pro-Ser-NH ₂ χ _yr _D-Ala -Phe-D-Ala-Tyr-D-Ala-SeT-NH ₂ Tyr-D-Ala-Phe-D-AIa-Phe-D-Ala-SeT-NH ₂ Tyr-D-Ser-Phe-D-Ala -Tyr-NH «Tyr-D-Ser-Phe-D-Ala-Phe-NH ₂

Some of these peptides have an even stronger opiate-like effect and / or have even greater stability compared to peptide-cleaving enzymes than the exemplary peptides described in Tab. 1 III and IV.

Biological effects of two exemplary peptides according to the invention .

Table 1 shows ^di: e pharmacological properties of two erfindungsgemäOer peptides compared to peptides of the prior art.

The guinea pig ileum preparation serves as a biological test system to demonstrate the opiate-like effect (for a detailed description, see DT-OS 29 21 216 ^ page 24). In this test those substance concentrations (nM) were calculated, which resulted in a 50 cent, inhibition of the electrically induced contraction of the organ preparation? (So-called IC, -. _N values). In addition, for a better understanding of the IC _n value. for the opiate normorphine and dermorphine (DT-OS 30 34 897) are set equal to 100% and the relative activity of the individual substances in comparison to normorphine or to dermorphine are given. The indication of the relative potency has the advantage that any fluctuations in the sensitivity of the test system are neutralized.

The values marked with (*) in Table 1 agree with those reported by de Castiglione et al. (Peptides, 2_, p. 266, Tab. 1, substances L and 3: column GPI = guinea p_ig Ji _- Ie um., Guinea pig intestine prepared 1901).

.-Substance

IC

50.

ReI. Value ReI. Value NQC = IOO. Of the. = 100

'Nor MQR-phin The morphine-Tyr-D-Ala-Phe-Pro-Tyr-NH ₂

Tyr-O-Ala-Phe-Gly-TyT-NH ₂

TyT-D-Ala-Phe-O-AIa-TyT-NH ₂

Ty-r-.D-Ala-Phe-D-AIa-PHe-NH ₂

110 5 * 100 3. 3143 100 110 7 * 1571 14th 5 786 2. 3667

3.2 100

3.5 50
25th
140 ·

TABLE 1 · '■

IC _5n values (nM), see text, of two exemplary peptides according to the invention (III and IV) compared to those of the prior art (dermorphin, I, II) measured on the isolated guinea pig ileum. The ICc _n values were averaged from 7 individual determinations; the standard deviation of a single value from the mean value is less than hk 14 «. The inhibitory effect of substances I, II, III and IV could be neutralized by adding the specific opiate antagonist Na.loxon · (final concentration in the organ bath 900 nM); if substances I to IV were further added to the organ bath (in the presence of naloxone), no further inhibition could be observed; the inhibition of substances I-IV is therefore to be regarded as opiate-specific.

Table 1 shows that both peptides III and IV according to the invention in comparison to peptide I of St. d. (29 36 099 DT-OS) are much stronger opiate ^ effective T, ie they have smaller IC, - _n values on. In comparison to peptide II of St. d. T. (DT-OS-30 34 897) also has a stronger opiate-like effect; Although the peptide III according to the invention has no higher activity than the "Pept.id II of St. dt", it is, as described in the exemplary embodiment, much more stable to peptide-cleaving enzymes (than II) and thus better for in vivo administration to achieve pharmacological ones Effects suitable.

The peptides according to the invention show strong opiate-like effects (analgesia) not only in vitro but also in vivo. The electrically stimulated rat tail served as the test system. The method was used as described by ßrantl et al. 1981 (Life Sciences, 28 , 1903-1909). The substances were administered intracerebroven.tricularly. The relative effectiveness in vivo (analgesia) of the substances in Table 1 (with comparative

substance dermorphin) corresponded roughly to the relative Potency determined using the isolated guinea pig ileum.

Enzymatic stability of the invention Peptides

1 mg each of the peptides dermorphin, I, II, III and IV (Tab. 1) were dissolved in 1 ml of freshly prepared Krebs-Ringer solution and each with 50 μg of OC-chymotrypsin (Serva, Heidelberg; in 50 μΐ Krebs-Ringer solution dissolved) mixed; Immediately after mixing, 100 μ.1 were removed, immediately heated to 95 ° C. and kept at this temperature for minutes (the heating served to inactivate the enzyme). The sample taken at the beginning of the incubation serves as the starting value (O value). The remaining 950 μΐ of the peptide / enzyme mixture was then washed 5 hours at 37 ⁰ C incubation; the incubation process was then also terminated by heating to 95.degree.

After centrifugation, the same aliquots of the respective supernatants were quantitatively collected their opiate-like effect, examined in comparison to the 0 value; as a verification system for the The guinea pig ileum again served opiate-like effects (as previously described). It was found that the opiate-like effect in the incubation batches with dermorphin

and the peptide II compared to the O value after a 5 hour incubation period almost had completely disappeared, whereas peptides III and IV had their full opiate activity maintained, i.e. that inhibition of the MeeTschweinchenileum preparation that was obtained by an aliquot of the O value corresponded to the same inhibition that was generated by an aliquot of the same volume of the 5-hour incubation mixture. Substance I shows greater stability than substance II, but is also essential less effective opaque than the peptides III and IV according to the invention. Overall, it can be seen that the introduction of a D-amino acid at position 4 of the known peptides I and II particularly advantageous for achieving a particularly strong opiate-like effect is and / or to obtain peptides that are particularly have high stability towards peptide-cleaving enzymes; Peptides which are used to achieve pharmacological effects are administered are subject, as is well known, to a very rapid metabolism, by peptide-cleaving enzymes which severely limit their uses as pharmaceuticals.

140 mg 480 mg 20th mg 10 mg 10 mg

Example of a pharmaceutical formulation (Tablet)

Substance III (Tab. 1)

Lactose

Cornstarch

Polyvinylpyrrolidone (PVP)

Magnesium stearate

The active ingredient comes along with lactose and corn starch mixed and granulated with a solution of PVP (50% in aqueous ethanol). The granules is dried; after the addition of magnesium stearate, the mixture becomes a tablet in the usual way pressed.

Claims (10)

Expectations 1) / Pharmacologically active / e peptides of the formula: • L-Tyr-XL-Phe-XT
L-Tyr-XL-Phe-XAT
L-Tyr-XL-Phe-XAXT
L-Tyr-XL-Phe-XAXBT where L-Tyr for the N-terminal amino acid residue L-tytosine, X for any amino acid residue of the D-configuration, L-Phe for the amino acid residue L-phenylalanine. -A and B can be any amino acid residues. T stands for OH, OR, NH ₂ , NHR, NR_ or NHNHR ', where R optionally has the following meaning: Substitute linear or branched C., _ ₁₀ -alkyl, adamantyl, C., -Cycloalkyl or ^ ₆ _ _Q ~ aralkyl, expediently phenyl, benzyl or phenylethyl and R 'denotes hydrogen, linear or branched C., "- alkyl, cycloalkyl or C, _fl -aralkyl, C _? "-Alkenyl, linear, branched or eyclic aliphatic. C,., - acyl, optionally substituted by OH, NH_, C.. -Alkoxy or halogen, aromatic acyl, optionally substituted by OH, NH ^, halogen or C,. -Alkoxy; linear branched or eyclic C-. ., aliphatic urethane or aromatic urethane and their pharmaceutically acceptable salts ;. 2) Pharmacologically active peptides according to claim 1, characterized in that the amino acid residue X by D-alanine, D-threonine, D-serine, O-methionine, D-valine, D-phenylalanine, D-leucine, D-isoleucine, D-arginine, D-histamine, D-proline, D-hydroxyproline, D-lysine, D-glutamine, D-glutamic acid, D-asparagine, D-aspartic acid and A, B by the aromatic amino acids tyrosine or phenylalanine, or glycine are replaced. 3) Pharmacologically active peptides according to "Claim 1 and 2, characterized in that a) the N-terminal L-tyrosine of the general Formula: NHW is present, where in detail: R, for hydrogen or an alkyl group with 1 to A C atoms, R. for hydrogen or together with R, for an ethylene bridge, Rc for hydrogen, an alkyl group with 1 to Carbon atoms or a R.CO group, R>. for a saturated or unsaturated, straight chain or branched alkyl radical with 1 to 17 carbon atoms, a phenyl radical or a Phenylalkyl radicals with 7 to 12 carbon atoms, the phenyl radicals being denoted by L oc) c; r 2 Su bn tuen from the series halogen, alkyl with I Ii ι:; 4 C- Atoms or alkoxy substituted with 1 to A C atoms may be, where the R-Q group 'is in the meta or para position to the -CH ₂ -C-CO-ReSt located, NHW W for hydrogen, alkyl with 1 to 5 carbon atoms, alkenyl with 3 to 5 carbon atoms, cyclopropylmethyl, cyclobutylmethyl, R ₆ CO-, b) the phenylalanine of the general formula is present C -CO - where in detail mean: R ₇ for hydrogen or alkyl with 1 to 4 C- Atoms,
R represents hydrogen, fluorine, chlorine, bromine, nitro, alkyl with 1 to k carbon atoms, ζ represents 1 or. 4) Pharmacologically active peptides according to one of the claims 1-3, characterized in that the amino acids alanine, phenylalanine and A and B as dehydroamino acids are present. 5) pharmacologically active peptides according to claims 1-4, characterized in that the phenylalanine in the 3rd amino acid position of the peptide (from the N-terminal End of calculation) is in the D-form. 6) Pharmacologically active peptides according to any one of claims 1-5, characterized in that this powerful effects on the central nervous system exercise. 7) Pharmacologically active peptides according to claim 1- 6, characterized in that the peptides induce strong opiate-like effects in vivo and in vitro, i 8) Pharmacologically active peptides according to claims 1-5, characterized in that they have strong effects exercise on the cardiovascular system. 9) Pharmaceutical preparations that act as the active ingredient one or more of those in the claims 1-0 contain peptides described. 10) Pharmacologically active peptides according to claims 1-3, characterized in that the peptides have the following structure: Tyr-D-Ala-Phe-D-Ala-Tyr-NH ₂ Tyr-D-Ala-Phe-D-Ala-Phe-NH ₂ Tyr-D-Ala-Phe-D-AIa-Tyr-D-Pro- NH ₂ Tyr-D-Ala-Phe-D-Ala-Phe-D-PTo-NH ₂ Tyr-D-Ala-Phe-D-Ala-Tyr-D-Pro-Ser-NH ₂ TyT-D-AIa-PhB-D-AIa-TyT-D-AIa-SeT-NH ₂ Tyr-D-Ala-Phe-D-Ala-Phe-D-AIa-SeT-NH ₂ Tyr-D-Ser- Phe-D-Ala-Tyr-NH «Tyr-D-Ser-Phe-D-Ala-Phe-NH ₂